Regulation of Apoptosis by Phosphatidylinositol 4,5-Bisphosphate Inhibition of Caspases, and Caspase Inactivation of Phosphatidylinositol Phosphate 5-Kinases

Abstract

Phosphoinositides such as phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate promote cell survival and protect against apoptosis by activating Akt/PKB, which phosphorylates components of the apoptotic machinery. We now report that another phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP 2 ) is a direct inhibitor of initiator caspases 8 and 9, and their common effector caspase 3. PIP 2 inhibited procaspase 9 processing in cell extracts and in a reconstituted procaspase 9/Apaf1 apoptosome system. It inhibited purified caspase 3 and 8 activity, at physiologically attainable PIP 2 levels in mixed lipid vesicles. Caspase 3 binding to PIP 2 was confirmed by cosedimentation with mixed lipid vesicles. Overexpression of phosphatidylinositol phosphate 5-kinase ␣ (PIP5KI␣), which synthesizes PIP 2 , suppressed apoptosis, whereas a kinase-deficient mutant did not. Protection by the wild-type PIP5KI␣ was accompanied by decreases in the generation of activated caspases and of caspase 3-cleaved PARP. Protection was not mediated through PIP 3 or Akt activation. An anti-apoptotic role for PIP 2 is further substantiated by our finding that PIP5KI␣ was cleaved by caspase 3 during apoptosis, and cleavage inactivated PIP5KI␣ in vitro. Mutation of the P 4 position (D279A) of the PIP5KI␣ caspase 3 cleavage consensus prevented cleavage in vitro, and during apoptosis in vivo. Significantly, the caspase 3-resistant PIP5KI␣ mutant was more effective in suppressing apoptosis than the wild-type kinase. These results show that PIP 2 is a direct regulator of apical and effector caspases in the death receptor and mitochondrial pathways, and that PIP5KI␣ inactivation contributes to the progression of apoptosis. This novel feedforward amplification mechanism for maintaining the balance between life and death of a cell works through phosphoinositide regulation of caspases and caspase regulation of phosphoinositide synthesis.Phosphoinositides have major roles in intracellular signaling and cell proliferation. The D3 phosphorylated phosphoinositides, phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) 1 and PI(3,4)P 2 , have been clearly implicated in the promotion of cell survival. They stimulate the phosphorylation of Akt/PKB (1), a serine/threonine kinase that inactivates multiple components of the apoptotic machinery (2-4). The D4 phosphorylated phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP 2 ), has not been directly shown to promote cell survival, although it may contribute in a number of ways indirectly. PIP 2 is a substrate for phosphoinositide 3-kinase that synthesizes the prosurvival D3 lipids (5), and it is a bona fide signaling molecule that regulates the actin cytoskeleton, vesicular trafficking, channel and transporter activities, and nuclear functions (6). PIP 2 synthesis is increased by growth factors (7), by thrombin (8), and by integrin signaling (9). In addition, PIP 2 inhibits gelsolin, a caspase substrate (10) that is a major effector of cytoskeletal changes (11). Recently...

PI4P5-Kinase Iα Is Required for Efficient HIV-1 Entry and Infection of T Cells

“…HIV-1 Env-mediated PIP 2 production during viral entry might therefore alter plasma membrane fluidity or viral receptor organization to favor initial HIV-1/cell interactions and promote subsequent fusion. PIP 2 has also been found to protect against cell apoptosis, alone or in a complex with gelsolin, by directly inhibiting the initiator caspases 8 and 9, as well as by binding and inhibiting their common effector, caspase 3 (37,38). Therefore it is plausible that HIV-1-triggered synthesis of PIP 2 during the first steps of the viral cycle may serve to ensure cell viability and, therefore, efficient viral infection and propagation.…”

Hypertonic Stress Increases Phosphatidylinositol 4,5-Bisphosphate Levels by Activating PIP5KIβ

“…PIP5KI Overexpression-We use the isoform designation for human PIP5KIs, which is different from that for mouse (30). Hemagglutinin (HA)-tagged PIP5KI␣, -␤, -␥87, and -␥90 were cloned from cDNA provided by other laboratories.…”

PIP5K-driven PtdIns(4,5)P2 synthesis: regulation and cellular functions

“…The depletion of PtdIns(4,5)P 2 in response to stress seems to be the result of the translocation of PIP5K away from the plasma membrane, which also occurs independently of caspase activation (Halstead et al, 2006). Other studies have indicated that PIP5K can be cleaved by caspase-3 (Mejillano et al, 2001). It is therefore possible that caspase-independent inhibition of PIP5K might initially induce a reversible decrease in PtdIns(4,5)P 2 levels, whereas longer-term caspase-dependent cleavage of PIP5K maintains the depletion of cellular PtdIns(4,5)P 2 levels.…”